This invention relates to radioimmunoassay methods and, more particularly to radioimmunoassay methods, reagents and packaged test kits for the in vitro determinations of L-triiodothyronine and thyroxine in unextracted blood serum.
Since the discovery in 1952 of L-triiodothyronine (3,5,3'-L-triiodothyronine or T3), this naturally occurring hormone has received attention because of its greater biological potency than thyroxine (T4) even though L-triiodothyronine is present in much smaller concentrations in blood serum than T4. It has been recognized for some time that T4 constitutes about ninety percent of the organic iodine-containing substances in the blood. After the initial demonstration of T4 in plasma, T3 was identified as the second circulating iodo-amino acid, and it has been shown that T3, like T4, is a normal thyroidal secretory product. The ease with which T3 and T4 are labeled with radioactive iodine has allowed for many studies of the detailed kinetics of metabolism of these compounds in man as well as experimental animals. Notwithstanding its low level in blood serum relative to T4, it has been estimated that T3 contributes a major portion of the calorigenic potency of the thyroid hormones. It has even been suggested that T3 is the active thyroid hormone and that T4 serves merely as a precursor or pro-hormone. In any event, the determination of T3 levels must be considered in the diagnosis of thyroid disorders.
The early measurements of T3 in plasma were by extraction and purification followed by either paper chromatography, gas-liquid chromatography or displacement analysis. Because of the formidable technical difficulties encountered and the discrepancies between the various methods, the values obtained were considered only tentative. In recent years, radioimmunoassay (RIA) methods have been developed for the direct determination of T3 in unextracted blood serum. These methods are based upon the principle of competitive protein-binding (CPB) with antibody as protein binder and make possible procedures with greater inherent specificity and sensitivity than that of other in vitro thyroid tests. In accordance with the competitive inhibition principle of radioimmunoassay, unlabeled or nonradioactive antigen (T3) in unknown blood serum samples competes against radioactive labeled antigen (T3) for binding to antibody and thereby diminishes the binding of the labeled antigen. In order to determine the concentration of T 3 antigen in an unknown sample, the degree of competitive inhibition observed in the unknown sample is compared with that obtained in known standard solutions.
As reported in the literature (Sekadde et al., Clin. Chem. 19/9, 1016-1021 (1973)), the known radioimmunoassay methods for determination of T3 depend upon the addition of a standard of unlabeled T3 or of an unknown solution to a fixed amount of T3 antibody followed by the addition of a fixed amount of radioactive labeled T3. An inhibitor to inhibit binding of T3 to thyroxine-binding globulin is also conventionally added to the mixture. The resulting mixture is typically incubated at 4.degree. C. for 16 to 72 hours following which the antibody bound T3 is separated from the unbound T3 by any one of a number of methods. In the radioimmunoassay method for T3 described by Sekadde et al., a buffered solution containing 8-anilino-1-naphthalene sulfonic acid (ANS), an inhibitor for inhibiting binding of T3 to thyroxine-binding globulin, is added to a series of tubes by pipetting. Each of a series of standard solutions containing known amounts of T3 is added to certain of the respective tubes and unknown serum samples are added to other tubes. T3-free serum is added to all tubes containing the standard solutions. Buffer solution, antibody and radioactive T3 are then added to the tubes and the mixture incubated at 37.degree. C. for 30 minutes. After cooling, a solution of polyethylene glycol is added to precipitate the antibody-bound T3 complex, the supernatant fluid is aspirated off and the precipitate counted with a gamma scintillation counter. The T3 value is then calculated as described. The authors state that the T3-free serum should be prepared weekly and that the buffered solution of 8-anilino-1-naphthalene sulfonic acid should be prepared daily.
Mitsuma et al. (Biochemical and Biophysical Research Communications, Vol. 46, No. 6, p. 2107-2113 (1972) describe a radioimmunoassay for the simultaneous determination of T3 and T4 in unextracted serum involving the sequential addition to glass tubes of unknown blood sample or standard T3-T4 solutions, solution of radioactive T3 and T4, inhibitor solution and antibody solution followed by incubation of the assay mixture for 90 minutes at 37.degree. C. After incubation, separation of antibody bound T3 and unbound T3 was carried out using a solution of dextran-charcoal and the resulting two fractions counted in a gamma counter.
Other radioimmunoassay methods for the determination of T3 in unextracted blood serum involving similar procedural steps are also reported in the literature. Hufner et al., Acta Endocrinologica, 72 (1973) 464-474; Hufner et al., Clinica Chimica Acta, 44 (1973) 101-107; Hesch et al., British Medical Journal, 1973, 1, 645-648 and Docter et al., Europ. J. Clin. Invest. Abstracts, Vol. 3, No. 3, (1973) 224-225.
While certain known radioimmunoassay methods for the determination of T3 in unextracted serum may be suitable for clinical use, their usefulness is somewhat limited because such methods are time-consuming and/or require a large number of procedural operations on the part of the technician which may introduce errors and affect the accuracy or reproducibility of the assay results. Thus, the commerically available test kits for use in carrying out T3 radioimmunoassay determinations typically contain a plurality of reagents and their clinical use requires the technician to perform many time-consuming operations in preparing the reagents and/or conducting the radioimmunoassay.
As mentioned, it has been conventional in the art to utilize an inhibitor for inhibiting binding of T3 by thyroxine-binding globulin, and the use of various inhibitors such as sodium salicylate, merthiolate, dilantin and tetrachlorothyronine has been reported in the literature. The use of 8-anilino-1-naphthalene sulfonic acid as an inhibitor was proposed by Mitsuma et al., supra. However, while this compound is a potent inhibitor of the binding of T3 by thyroxine-binding globulin and has been reported as the most effective inhibitor tested to date, it suffers from the serious drawback that it also inhibits antibody binding of T3. Hufner et al., Clinica Chimica Acta, 44 (1973) 101-107. Further, it has been found that 8-anilino-1-naphthalene sulfonic acid does not function as an effective inhibitor or yield reproducible results unless it is of the highest grade of purity.
Further, known methods for the radioimmunoassay determination of T4 have involved the use of antisera against thyroglobulin (Chopra, I. J. J. Clin. Endocr. 34:938 (1972)), T4-specific antibodies against conjugates of T4 and bovine serum albumin (BSA), human serum albumin (HSA) and ovalbumin (OA) (Menihold, H. and Wenzel, K. W., Horm. Metab. Res. 6 (1974) 169-170) and an antibody produced in response to injections of a T4-albumin conjugate (Dunn, R. T. and Foster, L. B., Clin. Chem. 19/9, 1063-1066 (1973)). However, it is desirable to have T4 antisera with greater specificity and higher avidity so as to provide greater sensitivity and improved reproducibility in the radioimmunoassay methods for the determination of T4.